Treatment of mercury vapor toxicity by combiningdeferasirox and deferiprone in rats

Marzieh Iranmanesh • S. Jamil A. Fatemi •

Mohammad Reza Golbafan •

Faezeh Dahooee Balooch

Received: 17 May 2013 / Accepted: 5 July 2013

� Springer Science+Business Media New York 2013

Abstract The hypothesis that combination of

deferasirox and deferiprone chelators might be more

efficient as combined therapy than single therapy in

removing mercury from the body was considered.

Male Wistar rats were exposed to mercury vapor for

2 weeks. After mercury administration some abnormal

clinical signs such as red staining around the eyes,

greenish mottling on the liver, weakness, loss of hair

and weight, were observed in animals. Chelators were

given orally after mercury vapor application for

2 weeks. Mercury toxicity symptoms in rats decreased

after drug administration. After chelation therapy,

these rats were anesthetized with ether vapor and

immobilized by cervical dislocation and then their

heart, liver, kidneys, intestine, spleen and testicles

were sampled for determination of mercury and iron

concentration. The combined chelation therapy results

showed that these chelators are able to remove mercury

from the body and toxicity symptoms decreased.

Keywords Deferasirox � Deferiprone �Mercury toxicity � Chelation therapy � Rats

Introduction

Mercury is a serious environmental pollutant with

toxic effects in all living organisms. Gold mining

emits elemental mercury vapor that is inhaled and

absorbed into the bloodstream (Grandjean et al. 1999).

Human exposure to inorganic mercury is mainly

occupational, which is often related to mining and

industrial activities. In addition, inorganic mercury is

believed to be the toxic species produced in tissues

after inhalation of mercury vapor. Because mercury

vapor generated from metallic mercury (Hg0) is highly

diffusible and lipid soluble, Hg0 vapor is absorbed

quickly and efficiently through the alveolar membrane

from inhaled air in the lung. Most inhaled Hg0 vapor is

oxidized rapidly to bivalent ionic mercury by catalase

in the blood and is distributed via the blood to various

organs (Magos et al. 1978). One way to remove toxic

elements from the body is chelation therapy. Chelation

therapy involves the use of ligating drugs that bind

metal for the treatment of potentially fatal conditions.

These ligands promote the excretion and subsequent

depletion of this transition metal in biological systems.

These chelating agents consist of a range of bidentate,

tridentate and hexadentate ligands in which two, three

or six atoms are able to coordinate, respectively

(Clarke and Martell 1992; Gomez et al. 1988). In this

procedure, chelators are administered orally in order to

remove toxic element. Deferasirox (4-[3,5-bis(2-

hydroxyphenyl)-1,2,4-triazol-1-yl]-benzoic acid, or

ICL670; Fig. 1a is a tri-dentate chelator with high

M. Iranmanesh (&) � S. J. A. Fatemi �M. R. Golbafan � F. Dahooee Balooch

Department of Chemistry, Shahid Bahonar University

of Kerman, 22 Bahman Blvd, Kerman, P.O. Box 76961,

Iran

e-mail: mzh_iranmanesh@yahoo.com

S. J. A. Fatemi

e-mail: fatemijam@yahoo.com

123

Biometals

DOI 10.1007/s10534-013-9656-9

selectivity for Fe3?. It selectively binds Fe3? over Fe2

? and shows little affinity for other divalent ions such

as Zn2? or Cu2? (Steinhauser et al. 2004). In vivo, this

selectivity is demonstrated by conserved plasma Zn

and Cu levels in patients taking deferasirox, and while

its efficacy is rather low for inducing negative iron

balance, it is effective and well tolerated (Nisbet-

Brown et al. 2003). In 2005 deferasirox became the

first FDA-approved oral alternative for treatment of

iron overload and was subsequently approved in the

EU in 2006 (Yang et al. 2007). Deferasirox possesses a

pFe3? value of 22.5 and can penetrate membranes

easily and possesses good oral availability. Indeed,

when orally administered to hypertransfused rats,

deferasirox promotes the excretion of chelatable iron

from hepatocellular iron stores four to five times more

effectively than desferrioxamine (Hershko et al.

2001). The other chelator for iron overload is deferi-

prone (1,2-dimethyl-3-hydroxypyrid-4-one) Fig. 1b.

Deferiprone is water soluble and can be given orally

(Hider et al. 1984; Kontoghiorghes et al. 1987;

Gyparaki et al. 1987). It possesses a pFe3? value of

20.5 and its important property is its ability to

penetrate cells, coordinate iron, forming a neutral

complex, which is also capable of permeating mem-

branes (Glickstein et al. 2006). At present, combina-

tion therapy with deferiprone and desferrioxamine,

that is highly selective for iron(III) under biological

conditions (pFe?3 = 26.6), is reported to be the most

effective treatment for many patients (Galanello et al.

2010). The combined therapy procedure is likely to

enhance iron excretion, target specific iron compart-

ments, minimize side-effects (by virtue of the use of

lower doses), facilitate individualization of therapy

and improve compliance (Ma et al. 2012). Desferri-

oxamine with a higher pFe?3 value acting as a sink.

Presumably deferasirox, also possessing a higher

pFe?3 value than deferiprone, behaves in a similar

manner. Recently successful chelation therapy using

both deferasirox and deferiprone has been reported

(Voskaridou et al. 2011). This kind of therapy by

combining two chelators is based on the assumption

that various chelating agents mobilize toxic elements

from different tissue compartments and therefore

better results are expected (Flora et al. 1995). Results

of this kind of combined chelation therapy have been

confirmed by (Amiri et al. 2007; Fatemi et al. 2007,

2009; Tubafard and Fatemi 2008). The aim of the

present research was to test the chelation potency of

deferasirox and deferiprone in combination, given to

animals after mercury loading. Testing was performed

by using a chronic poisoning model on rats with

individual and combined chelators given shortly after

mercury application.

Experimental

Apparatus

A Varian atomic absorption spectrometer (FAAS) was

used for measurement of mercury and iron concentrations

in various organs, respectively. Also a Mettler analytical

balance Model AE 160 was used in this research.

Maintenance of the animals

Male Wistar rats were obtained from Razi Institute

(Karaj, Iran). They were bred in animal house at

Kerman Neuroscience Research Center, Iran. The rats

were maintained under a controlled light:dark

(12:12 h) schedule at 23 ± 1 �C and the humidity of

55 %. The animals were assigned to control and

treated groups and were kept in well cleaned sterilized

cages. The rat food was purchased from Razi Institute.

This study was approved by the ethics committee of

Shahid Bahonar University of Kerman, Iran and

Kerman Neuroscience Research Center, Iran.

Materials

Mercury, deferiprone and other materials were pur-

chased from Merck Chemicals Co. and deferasirox was

purchased from Novartis Co. (Basel, Switzerland).

Fig. 1 Chemical structures of deferasirox (a) and deferiprone (b)

Biometals

123

Experimental design

In our research, the rats were classified as two groups:

control group and treated group. In control group,

inhalation of fresh air was carried out and in treated

group: they were exposed to mercury vapor. Exper-

iments were performed on 7-weeks-old Wistar male

rats.

There were slight differences between the groups in

the initial body weight of the rats (mean 225 g), but at

the end of mercury administration experiment, those

given mercury in their diet had significant weight loss

(Table 1). Comparison of the weights in this experi-

ment showed that dietary treatment affected the food

intake, whereby animals that were given normal diet

consumed more food than those given mercury. Also

because of the slight (but significant) differences in

body weight of rats at the start of the research, the

results can be influenced by the initial classification

and assignment of rats to treated groups. Therefore,

the day 1 groups’ body weights are notable and they

must be considered. Consequently after acclimatiza-

tion of the animals, we assigned them randomly to

control and treated groups.

Treated groups were exposed to mercury vapor for

2 weeks. Chelation therapy was carried out after

mercury application.

In this part of the research, treated groups were

divided into five groups: before chelation therapy,

without chelation therapy, chelation therapy with

deferasirox, chelation therapy with deferiprone and

chelation therapy with deferasirox ? deferiprone

(Table 2). Chelators (deferasirox and deferiprone)

were given orally after mercury application during

2 weeks. Doses of deferasirox and deferiprone were

140 and 300 mg/kg body weight, respectively.

Observed mercury toxicity symptoms in rats were

removed in short term after drug administration. After

chelation therapy, these rats were anesthetized with

ether vapor and immobilized by cervical dislocation

and their heart, liver, kidneys, intestine, spleen and

testicles samples were collected, weighed and dried

for determination of their mercury contents. The

samples were placed in an oven at 60 �C for 3 days.

They were then digested by 1.5 ml of HNO3 per 1 g of

dry weight tissues. After digestion, the solutions were

evaporated with the addition of 1.0 ml of H2O2 under

the hood. Then, the residue was diluted with water to

10 ml volume.

Statistical analysis

Determination of mercury and iron in samples were

carried out by FAAS. The values are expressed as

mean values (at least three separate determina-

tions) ± standard error of the mean. The data were

subjected to statistical analysis by Student’s t test;

P \ 0.05 was considered significant.

Results

Results of mercury raising and iron reduction in

organs of treated groups were statistically different.

A significant difference between control and treated

groups was observed. The general symptoms of

toxicity appeared after 2 weeks of mercury vapor

administration. Abnormal clinical signs in animals

were appeared as follows: darkening of the eyes,

yellowish discoloration of hair, flaccid, hypotonic

muscles, irritability, weakness and loss of hair. Also

the body weights of all animals in treated groups were

significantly decreased. The highest amount of

Table 1 Body weights over 2 weeks for the rats in different

groups (values are mean for the number of observation in

parentheses)

Group Control group Treated group

Initial body

weighta (g)

230 ± 6 (5) (day 1) 220 ± 4 (5) (day 1)

Final body

weighta (g)

245 ± 5 (5) (day 14) 225 ± 5 (5) (day 28)

a Mean of five determination ± standard deviation

Table 2 Classification of animals

All rats

Control

group

Treated group

Before chelation therapy

Without chelation therapy

Chelation therapy with deferasirox (140 mg/kg

body weight)

Chelation therapy with deferiprone (300 mg/kg

body weight)

Chelation therapy with deferasirox (70 mg/kg

body weight) ? deferiprone (150 mg/kg body

weight)

Biometals

123

mercury was found in the kidneys followed by

intestine. After the chelation therapy, the obtained

results showed that present mercury levels in all

tissues were significantly reduced and the symptoms

also decreased. There is statistical difference between

deferasirox and deferiprone in reducing the amount of

mercury in various tissues. The t test was applied to the

results assuming the certified values were the true

values. When comparing efficiencies of mono chela-

tors in this experiment, deferasirox was more efficient

than deferiprone in decreasing mercury concentration

in tissues. Comparison of mono and combining

chelators in this experiment show more efficiency of

deferasirox ? deferiprone in reducing the mercury

level in all tissues. The results of organ distribution of

mercury before and after chelation therapies for

mercury are shown in Table 3. Furthermore, iron

concentration after mercury administration remained

constant and after chelation therapy significantly

decreased thus consumption of iron tablet is recom-

mended to return iron level to its normal state. The

difference between iron values before and after

chelation therapy is notable. Combination of defer-

asirox ? deferiprone shows more efficiency in

decreasing iron level. The results of iron concentra-

tions before and after chelation therapies are summa-

rized in Table 4. In order to investigate the effect of

passing time in removing mercury from the body

spontaneously, one group was treated as without

chelation therapy. The results of chelation therapy

group are shown in Tables 3 and 4. Comparison of the

results obtained from both (before and without

Table 3 The results of

mercury levels before and

after chelation therapies

Five rats were placed in

each group

Results are represented as

arithmetic mean ± SEM

and are significant at

p \ 0.05 when compared

with control

Group Before

chelation

therapy

Without

chelation

therapy

Chelation therapy

with deferiprone

Chelation therapy

with deferasirox

Combination

therapy

Heart (mg/kg)

Control

group

0 – – – –

Treated

group

43.8 ± 1.4 42.1 ± 1.2 18.2 ± 1.1 14.8 ± 1.0 6.5 ± 0.5

Liver (mg/kg)

Control

group

0 – – – –

Treated

group

27.1 ± 1.6 25.1 ± 1.3 9.6 ± 1.0 7.5 ± 1.2 3.2 ± 0.9

Kidney (mg/kg)

Control

group

0 – – – –

Treated

group

113.1 ± 2.6 110.3 ± 2.3 51.7 ± 1.1 42.2 ± 1.2 19.5 ± 0.9

Intestine (mg/kg)

Control

group

0 – – – –

Treated

group

74.4 ± 1.6 69.1 ± 1.5 39.3 ± 1.1 26.1 ± 1.3 11.2 ± 0.8

Spleen (mg/kg)

Control

group

0 – – – –

Treated

group

27.2 ± 1.6 25.2 ± 1.3 9.6 ± 1.0 7.5 ± 1.2 3.2 ± 0.9

Testicle (mg/kg)

Control

group

0 – – – –

Treated

group

28.8 ± 1.2 26.1 ± 1.0 12.7 ± 1.1 9.2 ± 1.0 3.7 ± 0.4

Biometals

123

chelation therapy) groups indicate that the passing of

time has no significant effect on the removal of

mercury.

Discussion

The aim of the present research was to evaluate the

ability of deferasirox ? deferiprone in removing

mercury from rat organs. Many studies have now

reported the high absorption, distribution, long-term

efficacy and safety of deferasirox and deferiprone in

removing some toxic metal ions and treating iron

overload in patients with b-thalassaemia major (Cap-

pellini 2008; Neufeld 2006).

In this investigation, a short-term experimental

model was used in order to speed up the preliminary

testing procedure. The effects of these chelators on

mercury and iron levels were remarkable. It has been

reported that the chelating agents having higher

stability constants with a metal in aqueous solution

may also prove successful in reducing the body burden

of the metal (Kaur et al. 1984). Inhalation of mercury

vapor after exposure showed that the accumulation of

mercury in various tissues. In order to understand the

abilities of mentioned chelators, we have done the

distribution of mercury and observed accumulation of

direct toxic effect of mercury in rat organs. After the

administration of chelating agents, the mercury con-

tent reduced. A comparison of the results obtained

Table 4 The results of iron

levels before and after

chelation therapies

Five rats were placed in

each group

Results are represented as

arithmetic mean ± SEM

and are significant at

p \ 0.05 when compared

with control

Group Before

chelation

therapy

Without

chelation

therapy

Chelation therapy

with deferiprone

Chelation therapy

with deferasirox

Combination

therapy

Heart (mg/kg)

Control

group

6.5 ± 0.3 – – – –

Treated

group

6.5 ± 0.2 6.5 ± 0.4 5.1 ± 0.3 5.1 ± 0.2 4.7 ± 0.5

Liver (mg/kg)

Control

group

6.5 ± 0.2 – – – –

Treated

group

6.5 ± 0.1 6.5 ± 0.3 5.5 ± 0.2 5.1 ± 0.3 4.6 ± 0.4

Kidney (mg/kg)

Control

group

4.9 ± 0.2 – – – -

Treated

group

4.9 ± 0.1 4.9 ± 0.3 4.3 ± 0.2 4.1 ± 0.3 3.8 ± 0.4

Intestine (mg/kg)

Control

group

4.0 ± 0.2 – – – –

Treated

group

4.0 ± 0.1 4.0 ± 0.3 3.6 ± 0.2 3.5 ± 0.3 3.1 ± 0.4

Spleen (mg/kg)

Control

group

4.2 ± 0.2 – – – –

Treated

group

4.2 ± 0.1 4.2 ± 0.3 3.7 ± 0.2 3.6 ± 0.3 3.2 ± 0.4

Testicle (mg/kg)

Control

group

3.5 ± 0.2 – – – –

Treated

group

3.5 ± 0.1 3.5 ± 0.3 3.0 ± 0.2 2.6 ± 0.3 2.4 ± 0.4

Biometals

123

from with and without chelation therapies indicate

that combined (deferasirox ? deferiprone) therapy

increases the elimination of mercury from rat organs

effectively. Also toxicity and side-effects of deferasi-

rox and deferiprone are very low, therefore after basic

preclinical research, they could be recommended for

human administration. The important finding that

deferiprone leaves tissue iron levels close to normal is

fundamental and would suggest that the proposed use

of this chelator will not be highly toxic. The reason for

this important observation is that deferiprone is able to

redistribute iron in mammals (Evans et al. 2012).

In comparison to the results obtained by (Fatemi

et al. 2007, 2009; Amiri et al. 2007; Tubafard and

Fatemi, 2008) it can be also concluded that the two

chelators (deferasirox ? deferiprone) are more effi-

cient as combined therapy than single therapy in

removing mercury from rat organs. Therefore com-

bined therapy could eliminate mercury from rat organs

and treat side-effects and the general symptoms of

toxicity caused by mercury. Thus combination of

deferasirox ? deferiprone represent a promising drug

of mercury-mobilizing agent and could be recom-

mended for human administration.

References

Amiri A, Fatemi SJ, Fatemi SN (2007) Removal of thallium by

combining desferrioxamine and deferiprone chelators in

rats. Biometals 20:159–163

Cappellini MD (2008) Long-term efficacy and safety of defer-

asirox. Blood Rev 2:35–41

Clarke ET, Martell AE (1992) Stabilities of 1,2-dimethyl-3-

hydroxypyrid-4-one chelates of divalent and trivalent

metal ions. Inorg Chim Acta 19:57–63

Evans RW, Kong X, Hider RC (2012) Iron mobilization from

transferrin by therapeutic iron chelating agents. Biochim

Biophys Acta 1820:282–290

Fatemi SJ, Amiri A, Bazargan MH, Tubafard S, Fatemi SN

(2007) Clinical evaluation of desferrioxamine (DFO) for

removal of thallium ions in rat. Int J Artif Organs

30:902–905

Fatemi SJ, Tubafard S, Nadi B (2009) Evaluation of the effect of

cadmium on rat organs and investigation of diethyl car-

bamate as an oral drug in treatment of cadmium toxicity.

Med Chem Res 18:179–186

Flora SJS, Bhattacharyan R, Vijayaraghavan R (1995) Com-

bined therapeutic potential of meso dimercaptosuccinic

acid and calcium edentate on the mobilization and distri-

bution of lead in experimental lead intoxication in rats.

Fundam Appl Toxicol 25:233–240

Galanello R, Agus A, Campus S, Danjou F, Giardina PJ, Grady

RW (2010) Combined iron chelation therapy. Ann N Y

Acad Sci 1202:79–86

Glickstein H, BenEl R, Link G, Breuer W, Konijn AM,

Hershko C, Nick H, Cabantchik ZI (2006) Action of chelators

in iron-loaded cardiac cells: accessibility to intracellular labile

iron and functional consequences. Blood 108:3195–3203

Gomez W, Esparza JL, Domingo JL, Singha PK, Jones MM

(1988) Comparative aluminium mobilizing action of des-

ferrioxamine and four 3-hydroxypyrid-4-ones in alumin-

ium-loaded rats. Toxicology 130:175–181

Grandjean P, White RF, Nielsen A, Cleary D, de Oliveira Santos

EC (1999) Methylmercury neurotoxicity in Amazonian

children downstream from gold mining. Environ Health

Perspect 107:587–591

Gyparaki M, Porter JB, Hirani S, Streater M, Hider RC,

Huehns ER (1987) In vivo evaluation of hydroxypyridone

iron chelators in a mouse model. Acta Haematol 78:217–221

Hershko C, Konijn AM, Nick HP, Breuer W, Cabantchik ZI, Link

G (2001) ICL670A: a new synthetic oral chelator: evaluation

in hypertransfused rats with selective radioiron probes of

hepatocellular and reticuloendothelial iron stores and in iron-

loaded rat heart cells in culture. Blood 97:1115–1122

Hider RC, Kontoghiorghes G and Silver J (1984) Pharmaceu-

tical compositions. GB patent 2118176 A

Kaur G, Srivastava UC, Dwivedi RS, Srivastava RC (1984)

Influence of polyaminocarboxylic acids on the removal of

manganese-54 from the body organs of sham-operated and

partially hepatectomized rats. Toxicol Lett 22:1–6

Kontoghiorghes GJ, Aldouri MA, Hoffbrand AV, Barr J, Wonke

B, Kourouclaris T, Sheppard L (1987) Effective chelation

of iron in beta thalassaemia with the oral chelator 1,2-

dimethyl-3-hydroxypyrid-4-one. Br Med J 295:1509–1512

Ma Y, Zhou T, Kong X, Hider RC (2012) Chelating agents for

the treatment of systemic iron overload. Curr Med Chem

19:2816–2827

Magos L, Halbach S, Clarkson TW (1978) Role of catalase in

the oxidation of mercury vapor. Biochem Pharmacol

27:1373–1377

Neufeld EJ (2006) Oral chelators deferasirox and deferiprone

for transfusional iron overload in thalassemia major: new

data, new questions. Blood 107(9):3436–3441

Nisbet-Brown E, Olivieri NF, Giardina PJ, Grady RW, Neufeld

EJ, Sechaud R (2003) Effectiveness and safety of ICL670

in iron-loaded patients with thalassaemia: a placebo-con-

trolled, dose-escalation trial. Lancet 361:1597–1602

Steinhauser S, Heinz U, Bartholoma M, Weyhermuller T, Nick

H, Hegetschweiler K (2004) Complex formation of

ICL670 and related ligands with Fe(III) and Fe(II). Eur J

Inorg Chem 21:4177–4192

Tubafard S, Fatemi SJ (2008) Chelation of bismuth by com-

bining desferrioxamine and deferiprone in rats. Toxicol Ind

Health 24:235–240

Voskaridou E, Christoulas D, Terpos E (2011) Successful che-

lation therapy with the combination of deferasirox and

deferiprone in a patient with thalassaemia major and per-

sisting severe iron overload after single-agent chelation

therapies. Br J Haematol 154:654–656

Yang LPH, Keam SJ, Keating GM (2007) Deferasirox: a review

of its use in the management of transfusional chronic iron

overload. Drugs 67:2211–2230

Biometals

123